IGEM:MIT/2005/Friday 8/12 Meeting Agenda and Minutes

General Updates

• Biobricks request: please have up on wiki by end of today.
• Today: clean up in Kate's lab after lunch; spend rest time learning about lab space, doing lab duties, and organizing our stuff.
• Lets talk lab-use

SubTeam Updates

Format: what is the progress? what are the issues?

Input: Maxine

• Subtilis
  • Advantages
    • Is naturally competent (saves us time!)
  • Disadvantages
    • According to "The Permeability of the Wall Fabric of E. Coli and Bacillus subtilis,"
      • E. coli and Subtilis pore size: 25kDa
      • E. coli pore size (according to many other sources) 6Da
      • Our fluorescein constructs: ~8-16 kDa
    • The Com system in Subtilis degrades double stranded DNA and then resynthesizes one strand, so our fluorescein construct would not work
    • We need to relearn all lab techniques, i.e. plating, etc. because of its different properties, such as pore production
    • We need to remake much of the ToxR system (different promoters and ribosome binding sites) so that it can fit into subtilis
  • Conclusion: advantages not enough to outweight disadvantages at this point
• Most of the week was spent on helping FecA system

Receiver/Transmitter 1 - ToxR : Wiki-Will

• Activity:

-J07011 (ctx::gfp) might be correct, c/o gel test. Then again, it might not be. (see picture)

-J07009 is here! 
Initial assembly attempts - J13002::J07009, J07009::J07006, J07009::J07043, J07009::J07044, J07009::J07045
                    promotor, rbs :: ToxR     ToxR::malE      ToxR::scFv(i)   ToxR::scFv(ii)  ToxR::scFv(iii)
Every single plate of assemblies showed no growth this morning. I wonder what went wrong! can we back-track? 
my women's intuition tells me it was the transformation.

-Some troubling news shows up in our Gel Purify of J07009. The picture implies J07009 (cut x/p) is ~1kb, and it should bee ~650kb

• This Weekend (team effort):

- Retry Assemblies / retry transformation
- At the very least: miniprep large amounts of each biobrick being used right now.

Receiver/Transmitter 2 - FecA : Annie

• Updates

-QuikChange done on FecR, PhoA --> preparing to get them sequence
 FecA didn't get any colonies
-BB FecA Promoter
 However, gel on digest shows lots of other bands
-Recieve all primers reordered
-Ligating RBS & FecI
-Primers for PCR Tetracycline and Chloramphenicol resistance form hairpins and dimers
 Consistent with gel bands

• Questions

-Discuss the FecA Promoter gel bands with an advisor
-Only 1 internal primer or both forward & reverse for sequencing?
-Design new primers for PCR Tetracycline and Chloramphenicol? 
 Any other ways?

Actuator

No new updates.